Many facets of modern medicine and science depend on the ability to reliably measure the presence and quantity of compounds in solution. It remains difficult to differentiate a signal of interest arising from a single compound in a complex mixture of similar compounds. The problem is particularly severe in biological systems, as a molecule of interest in will often be part of a family of closely related compounds that appear similar (if not identical) to the detector.
Several techniques exist that can separate compounds out of a complex mixture so that they can be measured. Chromatography is a well-known example, wherein a small sample of the mixture is injected into a column of packed separation media-, and flushed through with an appropriate solvent. The compounds dissolved in the sample migrate down the column at different speeds and leave the far end at different times. However, the fractions collected at the end of the column inevitably have a much greater volume than the original sample. Dilution is the price paid for being able to tease the compound of interest away from its interfering relatives. While this tradeoff may be acceptable in many circumstances, when our samples are small, or the compound of interest is already dilute, the dilution of chromatographic separation is a serious problem.
Techniques to both isolate and concentrate solutes for analysis are rarely done because they are often cumbersome, multistep processes. They typically involve a separation step (e.g. chromatography with fraction collection) followed by a laborious concentration step, often involving evaporating or subliming away the solvent. It is seldom done because it costly, time consuming, requires trained technicians, and is difficult to do quantitatively.
Focusing techniques can perform both the isolation and concentration in one step. They most commonly depend upon the molecules in question being subjected to two opposing forces. For example, in gradient electrofocusing, the sample solution flows through a non-uniform electric field. The drag of the flowing fluid provides the first driving force on the molecules, and does not change over the length of the channel. The electric field drives the molecules against the direction of flow, and becomes stronger as they move down the channel. There is a point in the channel where the two driving forces become equal. This is where the molecules will become focused.
Focusing within a flowing channel has be demonstrated using a variety of methods, including electric field gradient focusing (EFGF), temperature gradient focusing (TGF) and isoelectric focusing (IEF). These lack a side channel to allow removal of the concentrated band. A variant of IEF, known as free-flow IEF, allows for a continuous separation (see Wen et al.). In the “free flow” format, focusing is performed perpendicular to the direction of flow. The degree of concentration is necessarily limited because solutes can only spend a finite time in the channel before being flushed out. There is no “trap-and-hold” capability.